Human serum albumin (hereinafter sometimes to be referred to as “HSA”) is widely distributed in the body, including blood and intercellular fluids. Its primary structure consists of 585 amino acids, and it is a simple protein having a molecular weight of about 66.5 kDa, which is free of a sugar structure. This protein is produced in the liver, mainly maintains normal osmotic pressure in the bloodstream, and is responsible for maintaining the liquid content of the blood. Therefore, HSA is used in various clinical situations for the treatment of a condition associated with loss of liquid from the blood vessel, such as surgery, shock, burn, hypoproteinemia causing edema and the like.
In addition, HSA functions as a carrier of various serum molecules, and is rich in safety, biocompatibility, biodegradation property, persistence in blood and the like. Therefore, it is considered a preferable carrier for a drug delivery system (DDS) of a drug having a problem in the kinetic property.
The DDS based on an irreversible bond between HSA and a drug includes a method improving persistence in blood of the drug bonded utilizing the long half-life of HSA, and a method using a modified form of HSA as a carrier of the active transport system. In the former, an attempt has been made to express a protein or a bioactive peptide having a short half-life as a hybrid by a gene fusion technique. In the latter, a method of controlling the physicochemical properties of HSA such as anionization and cationization, and an attempt to realize accurate kinetic control and cell-specific targeting by introduction of a recognition element (apparatus) of a receptor, which is present on the cell surface, such as sugar structure and peptide have been intensively studied (Lee Y C et al., Biochemistry, 15: 3956-3963, 1976, Opanasopit P et al., Am. J. Physiol. Gastrointest. Liver Physiol. 280: 879-889, 2001, Takakura Y et al., Biochemical Pharmacology 47: 853-858, 1994, Yamasaki Y et al., J. Pharmacol. Exp. Then 301: 467-477, 2002. Nishikawa M et al., Am. J. Physiol. Gastrointest. Liver Physiol. 268:0849-G856, 1995, Higuchi Y et al., int. J. Pharm. 287: 147-154, 2004).
It is known that a receptor that recognizes sugar residue and negative charge is present in the liver. Using this property, albumin bound with succinic acid, galactose, mannose and the like is used for targeting the liver.
However, for chemical modification of HSA, the following problems have been pointed out.
(1) The liver does not recognize unless very many sugar residues are bound;
Galactose-modified albumin is not recognized by the liver unless 10 or more galactoses are bound per albumin molecule (see Nishikawa M et al., Am. J. Physiol. Gastrointest. Liver Physiol. 268:G849-G856, 1995).
(2) The cell specificity to liver nonparenchymal cell is low;
Mannose- or fucose-modified albumin is known to be introduced into the both cells of liver endothelial cell and kupffer's cell (see Higuchi Y et al., Int. J. Pharm. 287: 147-154, 2004).
(3) A uniform bound form is difficult to prepare, and appropriate binding conditions need to be found; and the like. Accordingly, there is a strong demand for the development of a method for modifying HSA by a non-chemical technique.
It is an object of the present invention to provide uniform glycosylated albumin, particularly serum albumin, which specifically transfer to the liver, particularly kupffer's cell, thereby providing a drug carrier suitable for DDS to the liver.
The present inventors have conducted intensive studies in an attempt to solve the aforementioned problems and succeeded in preparing a glycosylated HSA with high liver transferability wherein a high-mannose type sugar chain is added to the Asn residue of a consensus sequence (Asn-X-Thr/Ser) by introducing the consensus sequence of an N-linked sugar chain into a DNA encoding HSA by site-directed mutagenesis, and cultivating Pichia pastoris transformed with an expression vector containing the obtained DNA encoding a mutant HSA, which resulted in the completion of the present invention.